Flow cytometric study of changes in the intracellular free calcium during the cell cycle

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Flow cytometric study of changes in the intracellular free calcium during the cell cycle
  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/14478331 Flow cytometric study of changes inintracellular free calcium during the cell cycle  Article   in  Cytometry · May 1996 DOI: 10.1002/(SICI)1097-0320(19960501)24:1<55::AID-CYTO7>3.0.CO;2-H · Source: PubMed CITATIONS 32 READS 21 3 authors , including:Narayana AnilkumarKing's College London 51   PUBLICATIONS   2,645   CITATIONS   SEE PROFILE Subramanian Manogaran PulicatKing Faisal Specialist Hospital and Research … 35   PUBLICATIONS   1,122   CITATIONS   SEE PROFILE All content following this page was uploaded by Narayana Anilkumar on 22 October 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.    zyxwvutsr 996 Wiley-Liss, Inc. Cytometry zy 4:55-63 (1996) Flow Cytometric Study of Changes in the Intracellular Free Calcium During zy he Cell Cycle Gopal Pande, N. zyxwv nil Kumar, and P.S. Manogaran Centre for Cellular and Molecular Biology, Hyderabad, India Received for publication July 31, 1995; accepted November 15, 1995 We measured the intracellular levels of free zyxw yto plasmic calcium in Werent phases of the cell cycle in viable rat fibroblasts, using two parameter flow cytometric analysis with Hoechst 33342 as the DNA specific dye and Fluo-3 as he calcium sensitive dye. We studied changes in calcium levels during the G1 phase of cell cycle by arresting cells with chemical agents such as stamsporine and hydmxyurea or by density dependent arrest of cell growth. We show that levels of calcium are lowest at the beginning of G1 phase but rise steadily with its progression and culminate at the Gl/S border. Our results suggest that complex changes occur in calcium levels dur- ing the process of mitotic division. During progres- sion of zyxw   and G2 phases, calcium levels decline and increase respectively. Our results offer a new meth- odology to estimate intracellular calcium levels in specific phases of the cell cycle. Based upon these results we propose a general scheme representing the changes in the intracellular calcium concentra- tion during the progression of the cell cycle. 1996 Wiley-Uss, hc. Key terms: Calcium, cell cycle, staurosporine, hy- droxyurea, nocodazole The role of calcium in regulation of extracellular and intracellular events has been well documented zyxwvu   1,2,4, 5,18,19,22,25,27). While in the extracellular compart- ment calcium ions play an important role in the binding of several growth factors to their specific receptors on cells, in the intracellular environment they play a central role as second messengers (19). Through the interplay of Ca2+ and several proteins, cells regulate key steps in the cell cycle such as reentry of quiescent cells into prolif- eration and the transition through the GUS, G2/M, and the metaphase/anaphase boundaries 1,4,16,27,28). Al- though the precise role of calcium in these transitions is not clear, changes in calcium levels do reflect the nature of the specific biochemical reactions that occur at these transitions (32). Flow cytometry is a powerful tool which allows the simultaneous measurement of several parameters of the same cell (7329). Using this technique on different cell types, researchers have demonstrated the expression of specific markers, such as p53/Rb protein (10,20), Ki67 antigen (17), cyclins (9,14), and also of nonspecific markers, such as total RNA and/or proteins 1 l), in dif- ferent phases of the cell cycle. The technique of multi- parameter cell cycle analysis has lead to some very inter- esting new ideas, one of which has been the delineation of the G1 phase into different parts, i.e., Glq, Gla, Glb, and Glc by using markers such as the total RNA content of cells and specific cyclin expression (9,14). The levels of intracellular calcium during the different stages of the cell cycle have not yet been studied as a continuous pro- cess, although a preliminary report is available (1 1). So far all studies have utilized cell lysis followed by micro- fluorimetry or other chemical methods for the measure- ment of intracellular calcium during the cell cycle (30, 31,33). These methods often do not reflect the hetero- geneity in calcium distribution among the cells at the same stage of the cell cycle. In this study we measured intracellular calcium in z i- able cells during the cell cycle of rat fibroblasts, by using Fluo-3 as the specific fluorescent dye for cytosolic “free- calcium,” and Hoechst 33342 for ATbases in DNA. This approach allowed us to study the dynamic changes as they occur during the passage of the same cells through different phases of the cell cycle. MATERIALS ND METHODS Cell Lines All the experiments were done with a rat fibroblast cell line F11 1 which was maintained in DMEM contain- ing 10% FCS and antibiotics (penicillin and streptomy- cin) in 5% CO, and 95% humidified air. Cells were sub- cultured after every 4 days, ix., before they became fully This work was partly supported by grants from the Department of Biotechnology, New Delhi. Address reprint requests to Gopal Pande, Centre for Cellular and Mo- lecular Biology, Uppal Road, Hyderabad, 500 007 lndia E-mail: gpande@ccmb.uunet.in.  56 PANDE zyxwvu T zyxwv L. confluent. All experiments were done within 10 passages after revival of the cells from liquid nitrogen storage. Chemicals All cell culture media and serum were from Gibco-BRL (Gaithersberg, MD, U.S.A.). Staurosporine, nocodazole, and hydroxyurea were from Sigma Chemical Company (St. Louis, MO). Fluo-3-AM and Pluronic F- 127 were from Molecular Probes (Eugene, OR, U.S.A.). All buffers were prepared from analytical grade chemicals available lo- Cell Synchronization in Gl/GO In order to arrest cells in early, middle, and late G1 phase, sub-confluent F1 1 1 cells were treated with stau- rosporine zyxwvutsr   12 nM and zyxwvut   nM) or hydroxyurea (3 mM), respectively, and incubated overnight. The possibility of these chemicals affecting the intracellular levels of cal- cium by their intrinsic effects was ruled out by short- term (1 5-30 minutes) treatment of cells with them and comparing the calcium levels with the untreated cells. For GO synchronization, the cells were allowed to grow to total confluency and kept as such for 4 days. After all the treatments, the cells were collected by mild zyxwv ryp- sinization (0.01% trypsin, 5 mM EDTA), were washed thrice in PBS, and stained. Collection of Mitotic and Interphase Cells Mitotic cells were prepared by a variation of the method described by Zieve et al.(34). After removal of the free floating cells by gentle shaking, subconfluent cultures were grown for zyxwvut   hours in nocodazole (1 pg/ ml). Then, the medium was gently aspirated and fresh, serum free medium (with 1 pwml nocodazole) was added. The mitotic cells were dislodged by tapping of the sides of the culture flask. The medium containing mitotic cells was collected and kept on ice. The remaining at- tached cells in the flask were harvested as described ear- lier. These interphase cells were washed several times with serum-free medium before use. The purity of mi- totic and interphase cell population was determined by staining the cells with acridine orange (25 pg/ml) and observing under a fluorescence microscope. Cell Staining The cell suspension (2 X lo5 cells) in 0.2 ml DMEM containing 10 serum, was chilled on ice and 0.4 ml of ice-cold solution A (0.1 Triton X-100, 0.08 N HC1, 0.15 M NaCI) was added, mixed well, and further incubated on ice for 15 zyxwvu   Then 1.2 ml of solution B (acridine orange 6 pglml in 1 mM EDTA and 150 mM NaCl prepared in phosphate-citric acid buffer, pH 6.0) was added to the cells. Fluorescence was measured within the next 3-15 min. Hoechst 33342 and Fluo-3 staining. The cell sus- pension in DMEM (2 X lo5 cells per ml) were stained with Hoechst 33342 (2 pglml) for 1 h at room temper- ature to stain DNA. The cells were then stained with Hue-3 by adding 1 pI of the stock solution (2 mM in DMSO) to 1 ml of the cell cally. Acridine orange staining. suspension with Pluronic F-127 as loading detergent (20% stock in DMSO, 0.1% final concentration) for 30 min. The cell suspension was washed thrice with DMEM and the cells were resuspended in medium containing Hoechst dye, for flow cytometric analysis. Ethidium bromide staining. The cells stained with HO 33342 and Fluo-3 were further stained with ethidium bromide (2 mg/ml) just before flow cytometric analysis. This staining was used to gate out the dead cells and thus ensure that only the viable cells are analyzed. Flow Cytometric Analysis Flow cytometry of live cells was performed on FACStar Plus (Becton Dickinson, San Jose, CA) equipped with two lasers (Coherent, Palo Alto, CA). A 6 W laser (Innova 90-6), tuned to emit 100 mW of zyx V light at 351-363 nm, was in the secondary position and a 5 W laser (Innova 90-5), tuned to emit 200 mW at 488 nm, in the primary position. Hoechst 33342 bound DNA fluorescence was studied by excitation with V and collecting the emitted fluores- cence through 400 nm Lp and 424 nm DF filters at the Fl-3 position; Fluo-3 fluorescence was measured by ex- citation at 488 nm and measuring the emitted fluores- cence through a DF 530/30 filter at the F1-1 position; ethidium bromide fluorescence was recorded at the F1-2 position using 488 nm as excitation wavelength and 630DF22 emission filters; acridine orange fluorescence was recorded using 488 nm as excitation wavelength and DF530/30 for DNA specific fluorescence and 630 DF22 for RNA specific fluorescence In all experiments, 20,000 events were recorded. In the supravitally stained cells, dead cells were gated out by displaying ethidium bromide fluorescence (Fl-2) and cell size (FSC); only the viable cells were analyzed for calcium or DNA. For the cells stained with acridine or- ange, gating of intact cells was done by FSC and side scatter (SSC) display. In all the experiments the cell via- bility was between 80 and 95%, and thus the data in all figures represent approximately 16,000-18,000 cells. Cell analysis was done using the Lysys I1 software (Bec- ton Dickinson). Depending upon the requirements, dual and single parameter analysis of cells was done using contour plots, dot plots and histograms. In order to distinguish fluorescence from clumps of cells versus fluorescence from single cells containing 4C DNA (G2 and M cells), “pulse processing” protocol was used and fluorescence from clumps of cells was gated out using the fluorescence-area (Fl-A) display. Calcium fluo- rescence of cells was measured by using logarithmic am- plification of the Fl-1 PMT; its estimation in G1 and S and G2 + M specific cells was done by gating the cells on the basis of DNA content (Hoechst 33342 fluorescence) and then displaying Ca2 + levels (Fluo-3 fluorescence), RESULTS Dual Parameter Analysis of DNA and Calcium With the aim of measuring the DNA content of live cells in relation to changes in levels of cytoplasmic Ca2+  CALCIUM IN CELL CYCLE zyxwv 7 11:~1~MITCA16B01 I 53% zyxwvuts op zyxwvu   F32-A\F32-Area > zyxwvutsrq ll:/l/MITCA16001\F32-A\F32-Area C {, G2+M m 0 --> 200 (DNA) 400 600 FIG zyxwvutsrqponmlk . Calcium, RNA, and DNA distribution in normal untreated rat fibroblasts. The cells were stained with Hoechst-33342 and Fluo-3 or acridine orange as described A shows calcium-specific Y axis) and DNA specific (X axis) fluorescence in normal untreated cells. The heteroge- neity of G1 cells with respect to calcium-specific luorescence is evident in the picture. B shows the RNA (X axis) and DNA (Y axis) specific F1 1 1 cells were simultaneously stained with Hoechst 33342 (a supravital stain for nuclear DNA) and nuo-3 (a calcium sensitive dye) and analyzed by flow cytometry as described. To ensure that the differences in the Fluo-3 fluorescence are not due to differences in the size of the cells, Fluo-3 fluorescence was analyzed against forward scatter; we found that the full range of calcium fluores- cence (from channels 70 to 600) is distributed through a uniform cell size on FSC axis (not shown). A typical display of DNA fluorescence and Fluo-3 flu- orescence is shown in Figure 1 (A, C, and D). Figure 1A shows DNA fluorescence (X axis) and Ca fluorescence (Y axis) simultaneously as a contour plot. The set of con- tours to the left represent GI cells which exhibit three levels of Ca2 specific fluorescence. These contours were used to set markers for the levels of calcium fluo- rescence (Fig. 1D). The three levels of Ca2+ fluores- cence-low, medium, and high-are shown by the mark- ers M1, M2, and M3, respectively, and the same levels are referred to in the subsequent figures. Our calculations showed that logarithmically growing untreated cells have 61 cells with low calcium, 19 with medium, and 20% with high calcium. The percentage of cells in different phases of the cell cycle was measured using the histo- gram profile of DNA (Figure 1C)-M1 represents G1 cells, M2 shows S phase cells, and M3 shows G2 M cells. DNA and RNA Contents of Cycling Cells To study the heterogeneity of cells in different phases we measured the DNNRNA contents of cells by acridine a fluorescence after acridine orange staining. The early, mid, and late G1 cells can be distinguished on the basis of RNA fluorescence. C shows the DNA distribution profile in the cell population. D shows the calcium fluorescence of the total cells against number of cells. The markers M1, M2, and M3 in C show the G1, S, and G2 +M cells, respectively, and in D show the low, medium, and high levels of calcium. orange staining (see Materials and Methods) and the re- sults are shown in Figure 1B. It shows the typical pattern for the RNA/DNA distribution in a proliferating cell pop- ulation with G1, s, and G2 M regions clearly defined on the basis of DNA fluorescence; this pattern has also been described earlier (7). RNA content (Fl-2 fluorescence) increased progressively in the G1 phase and on that basis it can be classified into early, mid, and late G1 phase; with the onset of S phase the RNA content dropped to a slightly lower level, where it was maintained through the remainder of S phase. During the G2 phase we saw fur- ther RNA synthesis and therefore an increase in RNA con- tent. In all three phases we observed a small percentage of cells with minimal RNA levels: these cells are the qui- escent cell populations derived from all three cell cycle phases and are called Glq, Sq, and G2q cells, respec- tively. Ca2+ Levels in G1 In order to get more information on Ca2+ levels in different phases of the cell cycle, we set gates on the three DNA content regions marked in Figure 2A (also shown in Fig. 1C) and examined Ca2+ fluorescence in each region, i.e., G1, S, and G2+M. Based upon the flu- orescence intensity, low, medium, and high levels of Ca2+ were identified (as shown in Fig. 1D). The percent- age of cells lying in each level is shown Table 1. This analysis showed that, in S phase and in G2+M phase, there are up to 10 fewer cells in the low calcium re- gion, thereby implying that the lowest levels of Ca2+ are  58 zyxwvusr ANDE zyxwvu T AL. ll:/l/MITCA16001 zyxwvutsr 11:~1/MITCAl~l\FLl-H\FLl-~ight zyx 5 8 z b- M3 1 GI CELLS u) J zyxw 0 d 6. I I A 288 488 Q .... 1 F32-WF32-Arca ---> e > 26ec CALCIUM 46 688 11:/l/MITCF116881\FLl-H\FLl-Height 11:~l/MITCAl68el~FLl-H\FLl-Hcight 1 S PHASE CELLS \D C 1 G2+M CELLS FIG. 2. Intracellular free calcium distribution in G1, S, and G2 ub populations. Regions R2, 3, and R4 n A represent the electronic gates used for the analysis of calcium distribution in each of G1 S, and G2 + M cell populations. B shows the calcium histogram of the gated G1 cells associated with a subpopulation of G1 cells. Similarly, the highest percentage of cells zyxwvuts 23 for G1 and 22 for S phase) in the high level of Ca suggests that the highest level of calcium2+ is seen at the G1 zyxwvuts S border. The per- centage distribution of cells in the different levels of cal- cium depended on the distribution of cells in cell cycle phases at the time of harvesting the cells specially for the untreated cells (for comparison, see the distribution in Figs. ID, 3C). For a finer analysis of intracellular free calcium ([Ca'+]i) in G1 phase, we used different protocols to arrest cells at different points in G1 and measured the DNA, RNA, and calcium levels. The results are shown in Figure 3; the pattern of fluorescence in different panels is shown in the margin. Panels A-C in Figure 3 show the data from control untreated cells; Figure 3C shows the basic pattern of calcium fluorescence in G1. Hydroxyurea Arrest When cells were treated with 3 mM hydroxyurea the percentage of cells in G1 phase and S phase increased from 53% to 63% and 16% to 23%, respectively (Table 1 ), suggesting that the hydroxyurea blockage of cells is in late G1 and the GUS boundary. The percentage of high calcium containing cells increased from 28% to 50% (Ta- ble 1). The residual lower level of calcium in these cells could be contributed by the cells in mid-G1 or in mid S-phase. Staurosporine Arrest Staurosporine has multiple arrest points in G1 depend- ing upon the concentration of the drug used (8,23). We u) 1 Q e n U M3 I and M1. M2 nd M3 re the markers used to calculate the percentage of cells in each group. C is the calcium histogram of S phase cells, and D shows calcium histogram of G2 + M cells. used two concentrations of staurosporine on F1 1 1 cells and found that 50 nM staurosporine arrested cells in early G1 as seen in panels G and H of Figure 3. By comparing the patterns in panels A and G we estimated that the percentage of cells in G1 rose from 56% to 71 . The effect of this arrest in early G1 on the intracellular cal- cium is shown in panel l of Figure 3 and also in Figure 4C. We estimated that the percentage of cells with low cal- cium increased from 42 to 62 (Table l). The per- centage of cells in high and mid level calcium was, how- ever, reduced. The use of 12 nM staurosporine caused a late GI phase arrest, as checked by the RNA/DNA staining (data not shown); and, correspondingly, the level of high calcium containing cells increased from 28 to 35% (Fig. 4B . Since this arrest point is earlier than the one affected by hydroxyurea, the level of calcium in stauro- sporine treated cells (as represented by the peak channel position) as well as the percentage of cells in the high level calcium is lower than for cells blocked at the Gl/S boundary by hydroxyurea Fig. 3F). Confluent Cells Cells were kept confluent for 96 h as described in Materials and Methods. This leads to the cessation of the progression of the cell cycle in these cells and they get blocked in different parts of G1. DNA analysis showed an increase in G1 cells from 56% to 76% (Table 1); these cells were arrested mainly in the early and middle G1 phases and about 15% cells in Glq (as seen in Fig. 3K). Calcium levels in these cells also show a corresponding change of pattern from the control cells; the percentage
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